Sage Journals: Discover world-class research

Abstract

Overexpression of Rabl3 is associated with some malignancies. However, their relationship with hepatocellular carcinoma remains unclear. In this study, the expression of Rabl3 in hepatocellular carcinoma cell lines, and four pairs of matched hepatocellular carcinoma tissues and their adjacent normal hepatic tissues were detected by quantitative reverse transcription polymerase chain reaction and western blot. In addition, the protein expression of Rabl3 was examined in 162 cases of hepatocellular carcinoma by immunohistochemistry. Rabl3 in hepatocellular carcinoma cell lines was elevated at both messenger RNA and protein levels, and the Rabl3 protein was significantly upregulated by upto 3.3-fold in hepatocellular carcinoma compared with the paired normal hepatic tissues. Immunohistochemical analysis revealed that overexpressions of Rabl3 were 80.2% in hepatocellular carcinoma. Rabl3 is expressed at significantly higher rates in hepatocellular carcinoma compared with adjacent normal hepatic tissue (p < 0.01). Statistical analysis suggested the upregulation of Rabl3 was significantly associated with lymph node metastasis, tumor thrombus of the portal vein, and advanced clinical stage (p < 0.05). Furthermore, we found that overexpression of Rabl3 in hepatocellular carcinoma cells could significantly enhance cell proliferation and growth ability. Conversely, silencing Rabl3 by small hairpin RNA interference caused an inhibition of cell proliferation and growth. Our studies suggest that the Rabl3 is a valuable marker of hepatocellular carcinoma progression and that the overexpression of Rabl3 plays an important role in the development and pathogenesis of hepatocellular carcinoma.

Keywords

Rabl3 hepatocellular carcinoma immunohistochemistry western blot quantitative real-time polymerase chain reaction

Introduction

Hepatocellular carcinoma (HCC) is characterized by significant morbidity and high mortality rates worldwide. HCC is the second leading cause of cancer-related mortality, and the incidence of HCC has been steadily increasing in coming years.^1–3 HCC has a poor prognosis as a result of a low detection rate at the curable stages and a high rate of recurrence. At the time of diagnosis, the majority of patients are already in the middle-advanced stage, and the 5-year survival rate is very low.^4–6 Although it is widely accepted that the etiology of HCC includes both genetic and environmental factors, the molecular mechanism of its development and progression remains poorly understood, and thus far, no specific signature of HCC gene expression has been reported to allow for patient-tailored therapy strategies.^7,8 Furthermore, prediction of clinical prognosis of HCC is still reliant on conventional pathologic variables, such as tumor size, tumor grade, and lymph node.^9,10 Hence, it is of great clinical value to further understand the molecular pathogenesis of HCC and to identify effective early markers for the diagnosis and prognosis of the disease as well as novel therapeutic targets.

The gene of Rabl3 is located on 3q13.3 and is composed of eight exons and seven introns. It is a member of the Rab subfamily, the largest group in the Ras superfamily of small guanosine triphosphatases (GTPases), which contains over 70 putative members in the human genome. Rab proteins may bind to guanosine triphosphate (GTP) or guanosine diphosphate (GDP) and possess intrinsic GTPase activity to control GDP/GTP conversion.^11–13 Genetic and functional studies on these Rab proteins, together with their associated regulators and effectors, have supported their putative roles in endosome formation, intracellular vesicular transport, and cytoskeleton formation.¹⁴ Recently, emerging evidence indicates a link between alterations in the Rab small GTPases and tumorigenesis.¹⁵ Li et al.¹⁶ have found that the novel human gene Rabl3 showed clear activity relating to paclitaxel sensitivity in breast cancer cells. Their results demonstrated that overexpression of Rabl3 could prevent tumor cells from undergoing paclitaxel-induced apoptosis and as a consequence elevate resistance to paclitaxel. Silencing of Rabl3 expression could increase paclitaxel-induced apoptosis in MDA-MB-231 cells.¹⁶ Zhang et al.¹⁷ found that Rabl3 was frequently overexpressed in lung cancer cell lines as compared with normal lung fibroblast cell lines. Knockdown of Rabl3 in lung cancer cells significantly enhanced cell death. Their results suggest that high expression of Rabl3 might inhibit cell death in non–small cell lung cancers (NSCLCs) via repression of mitogen-activated protein kinase 8/9/10 (MAPK 8/9/10)-mediated autophagy.¹⁷ As a result, overexpression of Rabl3 resulted in the enhancement of cell proliferation, inhibition of apoptosis, and paclitaxel resistance in human cancer cell lines.

Although published studies have suggested that Rabl3 expression is upregulated in some tumor, including breast cancer, NSCLCs, implicating the possibility of using Rabl3 as an indicator of carcinogenesis and progression; however, the expression dynamics of Rabl3 in HCC and their potential biological roles in the hepatocarcinogenesis have not been elucidated; the expression of these proteins and association with HCC pathogenesis have not been reported. Whether or not, their functions in HCC development and metastasis remain unknown.

In this study, we revealed that the expression of Rabl3 was upregulated and was also related to the tumor–node–metastasis (TNM) classification in HCC. Our study indicates that overexpression of Rabl3 may be a novel diagnostic biomarker in patients with HCC and may provide the basis for identifying new therapeutic targets.

Materials and methods

Ethics statement

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Clinical Research Ethics Committee of The Third Affiliated Hospital, Sun Yat-sen University. Written informed consents were taken from all the participants and ethical guidelines under Declaration of Helsinki were followed.

Cell lines

HCC cell lines, including HepG2, Hep-3B, HuH7, and normal hepatic cell line, LO2 were obtained from Institute of Cell Biology of Chinese Academy of Science (Shanghai, China). All cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA), 100 U/mL of penicillin or 100 µg/mL of streptomycin, and incubated at 37°C in a humidified incubator with an atmosphere of 5% CO₂.

Patients and tissue specimens

Specimens were obtained from archived, paraffin-embedded tissue sections from 162 patients with HCC at The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China between 1 January 2008 and 31 December 2009. A total of 162 normal liver tissue specimens were obtained from the periphery of the cancer site and utilized as controls. Patients did not have a previous history of chemotherapy or radiotherapy. Histopathological diagnosis of all specimens was confirmed by a trained pathologist. The patient cohort included 144 men and 18 women with a median age of 53 years (range = 19–72 years); well differentiated carcinoma: 37 cases, moderately differentiated carcinoma: 112 cases, and poorly differentiated carcinoma: 13 cases. According to the TNM system from the International Association for the Study of hepatic Cancer, 64 cases were classified as stage A, 11 cases were stage B, 67 cases were stage C, and 20 cases were stage D.

RNA extraction, reverse transcription, quantitative real-time polymerase chain reaction

Total RNA of tissues or cells was extracted using TRIzol Reagent (Sigma-Aldrich, St. Louis, MO, USA). First strand complementary DNA (cDNA) was synthesized from 500 ng total RNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative polymerase chain reaction (PCR) was performed in a 20 µL standard PCR reaction mixture prepared in duplicate using an Applied Biosystems 7900 Prism Real-Time PCR system and SYBR Premix Ex Taq (TaKaRa, Dalian, Japan), in accordance to the manufacturer’s protocol. The relative gene expression levels were calculated using the ΔCt method. Quantitative PCR primers were as follows: Rabl3, sense: 5′-TTGGGAGACTCAGGTGTTGGGAAA-3′, antisense: 5′-CAGTTGGCACCAAATCCCTGTTGA-3′.¹⁶ The glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific primers were also used as the internal control. GAPDH, sense: 5′-CTCATGACCACAGTCCATGC-3′, antisense: 5′-TTACTCCTTGGAGGCCATGT-3′.¹⁸

Vectors and retroviral infection

Rabl3-specific small hairpin RNAs (shRNAs; the target sequence: 5′-CAAGAGCAUAUCUACAATT-3′) were designed and synthesized by GeneChem Co., Ltd. (Shanghai, China).¹⁶ The Rabl3 overexpression plasmid, Rabl3-pcDNA3.1(+), was synthesized by Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). HepG2 cells were transfected with 2 µg of Rabl3 shRNA or Rabl3-pcDNA3.1(+) vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA; Thermo Fisher Scientific, Waltham, MA, USA).

Western blotting

Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer and centrifuged at 12,000 r/min at 4°C. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The samples corresponding to 20 µg of protein were resolved on a 10% denatured sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking non-specific binding sites for 1 h with 5% skim milk, the membranes were incubated with Rabl3 antibodies (1:2000 dilution; Abcam, Cambridge, UK) overnight at 4°C. Then, the membranes were washed with Tris-buffered saline with Tween (TBST) and incubated with a secondary antibody for 1 h. The protein bands were detected by enhanced chemiluminescence agent (Millipore, Billerica, MA, USA). Antibody of α-tubulin was used as the loading control.

Immunohistochemistry

Immunohistochemistry for Rabl3 expression was performed using the Novolink™ Polymer Detection Systems (Leica Biosystems Newcastle Ltd., Benton Lane, UK). All sections were routinely deparaffinized and rehydrated, and the sections were then rinsed in phosphate-buffered saline (PBS, pH = 7.4) and subsequently treated for antigen retrieval. After cooling to room temperature for 20 min, the sections were rinsed in PBS and immersed in 3% H₂O₂ for 5 min to block the endogenous enzymes. Then, the sections were incubated with normal goat serum at 37°C for 5 min to block non-specific antibodies. After incubation with the rabbit anti-human monoclonal Rabl3 antibody (1:150 dilution; Abcam), the sections were rinsed in PBS and incubated with Post Primary Block for 15 min. After washing with PBS, the sections were incubated with Novolink™ Polymer for 15 min. The sections were then rinsed with PBS and expression was visualized by incubating with 3,3′-diaminobenzidine and counterstaining with hematoxylin. Sections were dehydrated, rendered transparent, covered with coverslips, and sealed with neutral gum.

Tumor and adjacent normal tissue specimens were assessed by two independent pathologists. Rabl3 expression was localized predominantly in the cytoplasm. Sections were considered positive if the expression was detected in more than 10% of the cells in the tumor or normal tissue.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Cells were seeded on 96-well plates at initial density of (0.2 × 10⁴ per well). At each time point, cells were stained with 100 µL of sterile 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye (0.5 mg/mL, Sigma, St. Louis, MO, USA) for 4 h at 37°C, followed by removal of the culture medium and addition of 150 µL of dimethyl sulphoxide (Sigma). The absorbance was measured at 570nm, with 655nm as the reference wavelength. All experiments were performed in triplicates.

Statistical analysis

Data were analyzed using SPSS version 13.0. (Chicago, IL, USA). The relationship between the expression of Rabl3 and the clinicopathological parameters was evaluated by χ² analyses. A value of p < 0.05 was considered statistically significant.

Results

Upregulation of Rabl3 in HCC

Western blot analysis showed that Rabl3 protein was highly expressed in HCC cell lines, including HepG2, Hep-3B, and HuH7, nevertheless, it was weakly expressed in LO2 (Figure 1(a)). Corresponding to the upregulation of Rabl3 protein, real-time PCR results showed that all HCC lines displayed significantly higher, which was upto 12.9-fold of Rabl3 messenger RNA (mRNA) compared with the LO2 cell lines (Figure 1(b)).

Figure 1.

Expression of Rabl3 is elevated in HCC. (a) Expression of Rabl3 protein in normal human cell lines LO2 and HCC cell lines HepG2, Hep-3B, and HuH7. Expression levels were normalized with α-tubulin. (b) Quantification of Rabl3 mRNA in LO2 and HCC cell lines. Expression levels were normalized for GAPDH. Error bars represent standard deviations calculated from three parallel experiments. (c) Expression of Rabl3 protein in each of the HCC (T) and normal hepatic tissue (ANT) paired from the same patient by western blotting. (d) Quantification of Rabl3 protein in each of the primary HCC (T) and normal hepatic tissue (ANT) paired from the same patient by western blotting. Expression levels were normalized with α-tubulin.

To confirm whether the overexpression of Rabl3 is clinically correlated to HCC pathogenesis and development, comparative analysis of Rabl3 expression was conducted on four cases of paired HCC and adjacent normal hepatic tissue. The expression of Rabl3 protein was also discovered to be upregulated in all four HCC tissues compared with adjacent normal hepatic tissues using western blotting (Figure 1(c)). Furthermore, protein quantification demonstrated that three HCC tissues displayed much more than two-fold increase of Rabl3 protein compared with adjacent normal hepatic tissues (Figure 1(d)). It is noteworthy that the protein expression of Rabl3 in the HCC cells and clinical HCC tissues was correlated with the mRNA expression level, indicating that the upregulation of Rabl3 in HCC may be principally created by transcriptional upregulation.

Overexpression of Rabl3 in archived HCC tissues

In hematoxylin and eosin slices (HE), the HCC tumor cells have large and atypical nuclei and mitosis, which are different from those of normal hepatocytes, immune cells, and other cell types (Figure 2(a)). Besides, the Ki-67 index is higher in HCC but lower in normal hepatic tissues (Figure 2(b)). To further detect whether Rabl3 protein overexpression is associated with clinicopathological characteristics of HCC, 162 archived HCC tissues were examined by immunohistochemical staining. Immunohistochemical analysis revealed the expression of Rabl3 predominantly in the cytoplasm (Figure 2(c) and (d)). Rabl3 protein was detected in 80.2% (130/162) of HCC specimens. In contrast, Rabl3 was barely detectable or only marginally detectable and was 31.5% (51/162) in normal hepatic tissues. Rabl3 was found to be upregulated in HCC compared with normal esophageal tissues. There was a significant difference in Rabl3 expression between HCC and normal hepatic tissue (p < 0.01; Table 1).

Figure 2.

Positive expression of Rabl3 protein in hepatocellular carcinoma (HCC). (a) H&E staining. The nuclei of tumor cells are large and have atypia, which are different from those of adjacent normal hepatic tissues (H&E 20 × 10). (b) Ki-67 immunohistochemical staining: Ki-67 expression is observed in the nucleus and Ki-67 index is higher in HCC but lower in adjacent normal hepatic tissues (10 × 10). (c and d) Rabl3 immunohistochemical staining: Rabl3 expression is predominantly observed in the cytoplasm and is visualized as brown–yellow staining in HCC. The expression of Rabl3 is negative in adjacent normal hepatic tissue (c: 10 × 10; d: 20 × 10).

Table 1.

The expression of Rabl3 between HCC and adjacent normal hepatic tissue.

Groups	n	Rabl3
Groups	n	Negative	Positive (%)
HCC	162	32	130 (80.2)
Normal	162	111	51(31.5)

HCC: hepatocellular carcinoma.

There was a significant difference in the expression of Rabl3 between HCC and adjacent normal hepatic tissue (p < 0.01).

Relationship between the overexpression of Rabl3 and clinicopathological parameters in HCC

Statistical analyses were performed to detect the correlation between the expression of Rabl3 protein and the clinicopathological characteristics of HCC. Rabl3 expression was related to tumor thrombus of the portal vein, metastasis of the lymph node, and clinical staging in HCC (p < 0.05), whereas it was not associated with other clinicopathological parameters (p > 0.05; Table 2). Thus, the results supported that the overexpression of Rabl3 is associated with HCC clinical development.

Table 2.

Relationship between the expression of Rabl3 and clinicopathological parameters in HCC.

Parameter	n	Rabl3
		Negative (%)	Positive (%)	p
Sex
Male	144	27 (18.8)	117 (81.2)	0.364
Female	18	5 (27.8)	13 (72.2)
Age (years)
<50	97	21 (21.6)	76 (78.4)	0.459
≥50	65	11 (16.9)	54 (83.1)
Tumor size (cm)
<5	90	17 (18.9)	73 (81.1)	0.757
≥5	72	15 (20.8)	57 (79.2)
Tumor numbers
Single	116	21 (18.1)	95 (81.9)	0.402
Multitude	46	11 (23.9)	35 (76.1)
AFP (µg/L)
<400	98	17 (17.3)	81 (82.7)	0.341
≥400	64	15 (23.4)	49 (76.6)
HBsAg
Positive	135	80 (29.7)	189 (70.3)	0.436
Negative	27	13 (36.1)	23 (63.9)
Tumor thrombus
Have	69	6 (8.7)	63 (91.3)	0.002
No	93	26 (28.0)	67 (72.0)
Lymph metastasis
Have	33	2 (9.9)	31 (93.9)	0.027
No	129	30 (23.3)	99 (76.7)
Pathological grade
High	37	11 (33.9)	26 (70.3)	0.185
Moderately	112	18 (29.1)	94 (83.9)
Low	13	3 (30.6)	10 (76.9)
Hepatic function
Child A	106	21 (19.8)	85 (80.2)	0.754
Child B	36	6 (16.7)	30 (83.3)
Child C	20	5 (25.0)	15 (75.0)
Clinical stage
A	64	21 (32.8)	43 (67.2)	0.001
B	11	4 (36.4)	7 (63.6)
C	67	5 (7.5)	62 (92.5)
D	20	2 (10.0)	18 (90.0)

HCC: hepatocellular carcinoma; AFP: alpha-fetoprotein; HBsAg: hepatitis B surface antigen.

Deregulation of Rabl3 inhibits proliferation and activity of HCC cells

To further investigate the biological role of Rabl3 expression in HCC progression, HCC cell line HepG2 was established to stably overexpress Rabl3 (Figure 3(a)). Furthermore, the result of MTT assay revealed that Rabl3 infected HepG2 cells grew faster than the control by day 5 after plating (Figure 3(b)).

Figure 3.

Upregulation of Rabl3 enhances proliferation and tumorigenicity activity of HepG2 cells. (a) Ectopic expression of Rabl3 in HepG2 cells analyzed by western blotting. α-tubulin was used as a loading control. (b) Ectopic expression of Rabl3 stimulates HepG2 cell proliferation as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.

Furthermore, the impact of Rabl3 expression on HCC proliferation was evaluated in Rabl3 knockdown cells (Figure 4(a)). The depletion of Rabl3 expression caused significantly compromised viability in HepG2 cells (Figure 4(b)). Taken together, these results suggested that Rabl3 is very necessary for the proliferation of HCC cells and may be involved in HCC genesis and formation.

Figure 4.

Knockdown of endogenous Rabl3 inhibits cell growth and activity. (a) Knockdown of Rabl3 in specific shRNAs-transduced stable HepG2 cells. α-tubulin was used as a loading control. (b) Silencing endogenous Rabl3 inhibits cell growth as determined by MTT assays.

Discussion

Rabs are mostly noted for the key roles that they play in regulating the formation of the cytoskeleton and dynamic transportation. It is thought that Rabs could profoundly affect the proliferation, survival, and invasiveness of cancer cells.^19,20 Rabl3 gene is a member of the Rabs subfamily, which is highly expressed in cancer cells and has close relationships with the progression and aggressiveness of different types of cancer.^16,17 Recently, some studies have demonstrated that overexpression of Rabl3 is releated to the pathological processes, such as cancer cell survival, apoptosis, invasion, and metastasis. Rabl3 has been also found to be upregulated in several types of cancers, including breast cancer, and so on.^16,17 All these findings have confirmed that the overexpression of Rabl3 is associated with the occurrence and progression of cancer.

To investigate whether the upregulation of Rabl3 is also related to the progression of HCC, we performed studies to characterize the expression of Rabl3 in HCC cell lines and tissues. Our study revealed that Rabl3 is upregulated at both the mRNA and protein levels in HCC cell lines as well as in HCC tissue specimens. Furthermore, immunohistochemical analysis displayed that the expression of Rabl3 was 80.2% in HCC tissue specimens, while the expression was significantly lower in adjacent normal hepatic tissue (31.5%, p < 0.01). Moreover, statistical analysis showed that overexpression of Rabl3 correlates with portal vein tumor thrombus, lymph node metastasis, and clinical staging. The expression of Rabl3 was higher in portal vein tumor thrombus, lymph node metastasis, and clinical stage of C and D. In contrast, Rabl3 expression was unrelated to patient’s gender, age, number of tumors, tumor grade, liver function, and so on. Therefore, our results suggest that Rabl3 might play a role in the carcinogenesis, development, and progression of HCC.

Furthermore, we studied the gain or loss function of Rabl3 through its ectopic overexpression or Rabl3 knockdown by shRNA interference (shRNAi) in HCC cell lines. The result demonstrated that Rabl3 knockdown by shRNAi can inhibit HCC cell proliferation and migration. Taken together, our results not only suggest a potentially promising use of Rabl3 as a diagnosis indicator but also implicate a possible link between the biological function of Rabl3 and the pathogenesis of HCC.

It was studied to suggest that overexpression of Rabl3 can directly stimulate the proliferation of tumor cells, and may regulate the genesis and migration of cancer cells, and promote the growth of tumor. Furthermore, some studies displayed that Rabl3 is a component in the focal adhesion kinase/extracellular signal–regulated protein kinases 1 and 2 (FAK/ERK) signaling pathway and can stimulate the differentiation and proliferation of tumor cells by activating signaling pathways.^21,22 Thus, Rabl3 can play an important role in hepatocarcinogenesis.

In conclusion, upregulation of Rabl3 is significantly higher in HCC than in adjacent normal hepatic tissue and likely play an important role in the pathogenesis of HCC. The expression of Rabl3 may also be used as diagnostic and prognostic indicator of HCC.

Footnotes

Acknowledgements

Y.J. and D.T. designed the research; Y.P., Z.L., and Z.F. performed the research; X.H. and D.H. analyzed the data; and Y.P. and Z.L. wrote the article. Y.P. and Z.L. contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study was funded by the Science and Technology Program of Guangdong Province (No. 2014A020212155).

References

Tsujikawa

Masugi

Yamazaki

. Immuno histochemical molecular analysis indicates hepatocellular carcinoma subgroups that reflect tumor aggressiveness. Hum Pathol 2016; 50: 24–33.

Cai

Hou

Rao

. High expression of H3K27me3 in human hepatocellular carcinomas correlates closely with vascular invasion and predicts worse prognosis in patients. Mol Med 2011; 17: 12–20.

Dugum

Hanouneh

McIntyre

. Sonic hedgehog signaling in hepatocellular carcinoma: a pilot study. Mol Clin Oncol 2016; 4: 369–374.

Shimizu

Kanda

Sugimoto

. NRAGE promotes the malignant phenotype of hepatocellular carcinoma. Oncol Lett 2016; 11: 1847–1854.

Mao

Wang

. Combretastatin A-1 phosphate, a microtubule inhibitor, acts on both hepatocellular carcinoma cells and tumor-associated macrophages by inhibiting the Wnt/β-catenin pathway. Cancer Lett 2016; 380: 134–143.

Wachsmann

Peng

. Molecular imaging and therapy targeting copper metabolism in hepatocellular carcinoma. World J Gastroenterol 2016; 22: 221–231.

Borgas

Gao

Tong

. Potential role of phosphorylation as a regulator of aspartyl-(asparaginyl)-β-hydroxylase: relevance to infiltrative spread of human hepatocellular carcinoma. Liver Cancer 2015; 4: 139–153.

Hayashi

Shibahara

Misumi

. Histologic assessment of intratumoral lymphoplasmacytic infiltration is useful in predicting prognosis of patients with hepatocellular carcinoma. PLoS ONE 2016; 11: e0155744.

Han

Kim

. Treatment of hepatocellular carcinoma with portal venous tumor thrombosis: a comprehensive review. World J Gastroenterol 2016; 22: 407–416.

10.

Grandhi

Kim

Ronnekleiv-Kelly

. Hepatocellular carcinoma: from diagnosis to treatment. Surg Oncol 2016; 25: 74–85.

11.

Subramani

Alahari

. Integrin-mediated function of Rab GTPases in cancer progression. Mol Cancer 2010; 9: 312.

12.

Recchi

Seabra

. Novel functions for Rab GTPases in multiple aspects of tumour progression. Biochem Soc Trans 2012; 40: 1398–1403.

13.

Marlin

. Rab family of GTPases. Methods Mol Biol 2015; 1298: 1–15.

14.

Cherfils

Zeghouf

. Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev 2013; 93: 269–309.

15.

Szatmári

Sass

. The autophagic roles of Rab small GTPases and their upstream regulators: a review. Autophagy 2014; 10: 1154–1166.

16.

Wang

Zeng

. Evaluation of the novel gene Rabl3 in the regulation of proliferation and motility in human cancer cells. Oncol Rep 2010; 24: 433–440.

17.

Zhang

Sun

Luo

. High expression of Rab-like 3 (Rabl3) is associated with poor survival of patients with non-small cell lung cancer via repression of MAPK8/9/10-mediated autophagy. Med Sci Monit 2016; 22: 1582–1588.

18.

Tang

Lei

. MicroRNA-155-3p promotes hepatocellular carcinoma formation by suppressing FBXW7 expression. J Exp Clin Cancer Res 2016; 35: 93.

19.

Cheng

Mills

. Assay of Rab25 function in ovarian and breast cancers. Methods Enzymol 2005; 403: 202–215.

20.

Amillet

Ferbus

Real

. Characterization of human Rab20 overexpressed in exocrine pancreatic carcinoma. Hum Pathol 2006; 37: 256–263.

21.

Han

Shi

. PTPIP51, a novel 14-3-3 binding protein, regulates cell morphology and motility via Raf-ERK pathway. Cell Signal 2008; 20: 2208–2220.

22.

Luchetti

Betti

Canonico

. ERK MAPK activation mediates the antiapoptotic signaling of melatonin in UVB-stressed U937 cells. Free Radic Biol Med 2009; 46: 339–351.

The overexpression of Rabl3 is associated with pathogenesis and clinicopathologic variables in hepatocellular carcinoma